Polynucleotides encoding platelet endothelial cell adhesion molecule (PECAM-1) and fragments thereof

ABSTRACT

A glycoprotein, PECAM-1, and variants thereof can be obtained by expression in a transformed host cell of a polynucleotide coding for the glycoprotein or a variant polypeptide. PECAM-1 can also be isolated from cellular sources. An antibody specific for PECAM-1 or a PECAM variant can be produced via recombinant techniques, or can be obtained from a hybridoma.

This Application is a continuation of application Ser. No. 977,567,filed Nov. 17, 1992, abandoned, which is a divisional of applicationSer. No. 466,140, filed Jan. 19, 1990, now U.S. Pat. No. 5,264,554.

BACKGROUND OF THE INVENTION

The present invention relates to polypeptide molecules that can act asligands for proteins and other agents that bind platelet and endothelialcell membranes in vivo.

Cells of many types have surface proteins, called "integrins," that arerecognized by extracellular proteins, such as fibronectin, vitronectin,ostopontin, collagens, thrombospondin, fibrinogen and von Willebrandfactor (VWF), which affect the attachment of cells to theirsurroundings. Integrins which act as receptors for several of theseextracellular proteins have been identified with human plateletglycoprotein Ib (GPIa), which partially mediates VWF-dependent adhesionof platelets to exposed vascular endothelium; GPIIa, which correspondsto the β chain of the fibronectin receptor; and the heterodimer proteincomplex GPIIb-GPIIIa, which functions in platelets as a receptor forfibrinogen, for VWF in the absence of fibrinogen, for fibronectin andfor vitronectin.

An α-β chain configuration common to GPIIb and GPIIIa is typical ofmembers of the integrin family of receptors. Another group ofadhesion-promoting proteins, referred to generically as "cellularadhesion molecules" (CAMs), are classified with proteins encoded bygenes of the immunoglobulin gene superfamily. See, e.g., Williams &Barclay, Ann. Rev. Immunol. 6: 381-405 (1988), the contents of which arehereby incorporated by reference. Like other proteins associated withthe immunoglobulin (Ig) superfamily, CAMs share a common structure--theimmunoglobulin homology unit--that is characterized by an amino-acidsequence, approximately 100 residues in length, having a centrallyplaced disulfide bridge which stabilizes a series of anti-parallel βstrands into the so-called antibody fold. In particular, theimmunoglobulin homology unit comprises a conserved amino-acid sequenceGly-X-X-Val/Leu/Ile-X-Val/Leu/Ile-X-Cys/(35-55 aminoacids)/Asp-X-Gly-X-Tyr-X-Cys-X-Val/Ala. See Hunkapill-er & Hood, Nature323: 15 (1986).

Among the known CAMs are intercellular adhesion molecule-1 (ICAM-1),which mediates leukocyte adhesion by binding the integrin lymphocytefunction-associated molecule-1 (LFA-1); carcinoembryonic antigen (CEA);fasciclin II; CD4, a T-cell subset marker which is a component of thecellular receptor for HIV-I; neuronal cell adhesion (N-CAM), whichmediates adhesion of neural cells; myelin-associated glycoprotein (MAG),which is understood to function in myelination; lymphocytefunction-associated antigen-3 (LFA-3) and the major glycoprotein ofperipheral myelin (Po). In addition to the functional association of CD4with HIV-I, the role of CAMs as viral receptors is reflected in the factthat receptors for two picornaviruses, human rhinovirus and poliovirushave been identified, respectively, with ICAM-1 and a cell surfaceglycoprotein comprised of three immunoglobulin-like loop domains(compared to five such domains for ICAM-1).

Although CAM molecules fulfill important functions in vivo, no proteinobtained in substantially pure form from platelets has ever beenidentified, structurally or functionally, as a member of the CAM family.A characterization of this sort would be significant because of therole(s) such a protein, and variant molecules based thereon, would beexpected to play in basic cell surface recognition events involvingplatelets, including self-association of platelets (aggregation), withother blood cells and with endothelial cells which line blood vessels;and platelet adhesion to extracellular matrix components, for example,subendothelium exposed in the course of vascular trauma.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a purifiedform of a CAM protein, found on platelet and endothelial cells, inamounts which are practicable to recover.

It is also an object of the present invention to provide polypeptidemolecules that function, in CAM-like fashion, as receptors for proteinsand other agents that bind to platelet membrane proteins in vivo.

It is a further object of the present invention to provide means forproducing a protein, possessing the structural attributes associatedwith the immunoglobulin superfamily, that can be used to influence aplatelet-surface recognition event.

In accomplishing the foregoing objects, there has been provided, inaccordance with one aspect of the present invention, a compositioncontaining a recoverable amount of platelet/endothelial cell adhesionmolecule-1 (PECAM-1). In one preferred embodiment, the composition isPECAM-1 in substantially pure form, while in another preferredembodiment the composition contains is mature PECAM-1.

In accordance with another aspect of the present invention, a PECAMvariant has been provided that is selected from the group consisting ofa PECAM mutein and a molecule that corresponds to a portion of PECAM-1or that comprises a portion of PECAM-1 without being coincidenttherewith, where the molecule displays adhesion-promoting activity. In apreferred embodiment, the PECAM variant corresponds to or comprises anextracellular portion, but not a transmembrane portion or anintracellular portion, of PECAM-1. In other preferred embodiments, theaforementioned extracellular portion respectively, comprises aN-terminus Gln-Glu-Asn-Ser-Phe and has an amino-acid sequence of 574residues.

In accordance with yet another aspect of the present invention, apolypeptide molecule has been provided that has an amino-acid sequencewhich comprises a portion of the PECAM-1 amino-acid sequence, whereinthe polypeptide molecule (a) is not coincident with said PECAM-1amino-acid sequence and (b) possesses PECAM-1 activity. In a preferredembodiment, the polypeptide is between 4 and 100 amino residues long.

Also provided, according to a further aspect of the present invention,are an isolated polynucleotide molecule that encodes PECAM-1 and apolynucleotide molecule that encodes a PECAM variant. In one preferredembodiment, a polynucleotide molecule encodes a protein, preferablyhaving a polypeptide molecular weight of about 79 kilodaltons (kd), witha N-terminus Gln-Glu-Asn-Ser-Phe-Thr-Ile and, moreover, hybridizes underhigh stringency conditions with an oligonucleotide probe that iscomplementary to a portion, at least about 10 nucleotides in length, ofthe nucleotide sequence depicted in FIG. 1.

An oligonucleotide is that additionally provided that corresponds to oris complementary to a portion of a nucleotide sequence depicted in FIG.1 such that the presence of the oligonucleotide, or a transcriptionproduct thereof, in a cell expressing a DNA sequence which encodesPECAM-1 or a PECAM-1 variant, inhibits transcription or translation ofthe DNA sequence. In preferred embodiments, the oligonucleotidle eitheris RNA (and inhibits translation of the DNA sequence) or is DNA (andinhibits transcription of said DNA sequence).

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Unless otherwise indicated, the respective contents of the documentscited below are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the primary structure of a platelet membraneglycoprotein within the present invention (PECAM-1), along witch thenucleotide sequence encoding that primary structure. The hydrophobicputative signal peptide and transmembrane domain of the matureglycoprotein are underlined. Two naturally occurring Eco R1 sites areboxed. Cysteine residues, spaced approximately 50 amino acids apartthroughout the entire external domain of PECAM-1, are circled; these arethought to participate in disulfide-bond formation within individualimmunoglobulin homology units. Predicted glycosylation sites aredesignated with black triangles. A potential tyrosine phosphorylationsite at residue 686 is indicated with a closed circle. The TAG stopcodon is underlined in bold.

FIG. 2 is a line drawing schematically depicting PECAM-1, along withseveral other CAM molecules mentioned above, in relation to the cellmembrane (stippled region). The circles are representations ofindividual immunoglobulin homology units. According to theclassification of Williams, Immunology Today 8: 298 (1987), the depictedCAMs are made up of C2-type domains, with the exception of CEA, whichhas one V-type domain at the amino terminus. In fasciclin II and N-CAM,the hatched boxes proximal to the membrane represent fibronectin typeIII domains. Putative N-linked carbohydrate chains on PECAM-1 aresymbolized by a ().

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A platelet membrane glycoprotein has been discovered that is properlycharacterized as a CAM, according to the criteria discussed above. Themature protein, lacking a 27 amino-acid signal peptide sequence, has amolecular weight of about 130 kilodaltons (kd). Although this size is inthe same range as a number of known platelet membrane glycoproteins, thenew glycoprotein can be obtained in recoverable amounts, pursuant to thepresent invention, in a form such that the glycoprotein preparationmigrates as a single band on a silver stained SDS-PAGE gel("substantially pure form"). Preferably, the glycoprotein is in a formof sufficient purity to be usable in therapeutic contexts, includingthose discussed below.

The glycoprotein of the present invention is designatedPlatelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1) because it is aCAM found on both platelet and endothelial cell membranes. In matureform, PECAM-1 has a 711 amino-acid sequence (polypeptide molecularweight: 79,578 daltons) which is depicted, along with the aforementionedsignal peptide, in FIG. 1. PECAM-1 is glycosylated to the extent thatapproximately 39% of its molecular weight is attributable tocarbohydrate, and the mature protein has nine putative,asparagine-linked glucosylation sites, marked by black triangles inFIG. 1. All these sites are present in the glycoprotein extracellulardomain, which has 574 amino acids. PECAM-1 also includes a 19 amino-acidtransmembrane portion and a 118 amino-acid cytoplasmic domain. Thisarrangement of domains (see FIG. 2) is consistent with an orientationfor PECAM-1, within the plasma membrane, that is typical of otherintegral membrane glycoproteins.

A polynucleotide that contains a nucleotide sequence encoding PECAM-1 isalso shown in FIG. 1. The sequence of 2557 base pairs (bp) contains a141-bp 5' untranslated (UT) region, a 2214-bp open reading frame whichcodes for the 738 amino acids that make up the mature protein plussignal peptide, and a 3' UT region of 202 bp. To obtain a polynucleotidewhich contains a PECAM-1-encoding sequence, it is preferable to use thesequences of the 5' and 3' UT regions to construct complementaryoligonucleotide primers, in order to amplify the PECAM-1 coding sequencevia polymerase chain reaction (PcR) or similar amplification technique.

PECAM-1 can be found in the membranes of neutrophils, monocytes, bonemarrow cells, immature lymphoid cells, myeloid hematopoietic cells andcells from leukemia and lymphoma patients, as well as in platelet andendothelial cell membranes. Substantially pure PECAM-1 can be producedin recoverable amounts by isolating the protein from these cellularsources, and by means of conventional genetic-engineering techniqueswhich are discussed in greater detail below.

A "recoverable" amount in this regard means that the isolated amount ofthe glycoprotein is detectable by a methodology less sensitive thanradiolabeling, such as an immunoassay, and can be subjected to furthermanipulations involving transfer of the protein per se into solution.Preferably, a recoverable amount of PECAM-1 should be such thattransferring the protein into solution yields a concentration of atleast 50 nM, and more preferably at least 1 μM.

Isolating recoverable amounts of substantially pure PECAM-1 from naturalsources preferably involves the preparation of solubilized membraneproteins from PECAM-1-containing cells, such as platelets, andpurification of the mature glycoprotein via immunoaffinitychromatography according to known procedures. See Wilcheck, et al.,Meth. Enzymol. 104: 3 (1984); and Ausubel, et al. (eds.), CURRENTPROTOCOLS IN MOLECULAR BIOLOGY Wiley Interscience, New York (1987,1989),at Section 10.11. For example, platelets can be isolated, solubilizedand centrifuged, followed by application of the resulting supernatant toa lectin-sepharose column, e.g., a Conconavilin A (ConA)-sepharosecolumn. Since PECAM-1 does not bind to ConA-like lectins, theflow-through of such a column can be used to obtain a PECAM-1-enrichedpreparation.

For further purification, PECAM-1-specific monoclonal antibodies (MAbs)can be attached to an affinity column, such as a sepharose column (see,e.g., Ausubel supra, at Section 10.16), and the flow-through mentionedabove can be refluxed through the affinity column to bind PECAM-1.Nonspecific binding can be diminished during the PECAM-1 binding byadjusting pH and salt concentration in the column. Substantially purePECAM-1 can then be eluted from the column using a suitable acidicelution buffer containing a mild detergent, such as digitonin,octylglucoside, CHAPS or glycine/Triton X-100, and then recovered inuseful amounts.

The purity of the PECAM-1 thus obtained can be assessed by running thePECAM-1 preparation on SDS-PAGE, under reducing and nonreducingconditions, to determine whether the PECAM-1 runs as a single band. Ifnecessary, the eluted PECAM-1 can be adsorbed to an additionallectin-sepharose column, such as wheat germ sepharose, and eluted offwith a suitable elution buffer, e.g., a glycine buffer containing a milddetergent, to obtain substantially pure PECAM-1.

Antibodies suitable for use in the immunoaffinity-chromatographicpurification of PECAM-1 can be obtained as described in greater detailbelow. It has also been discovered that certain MAbs employed previouslyin other contexts, such as for distinguishing different stages oflymphoid and myeloid differentiation, can also be used in theimmunoaffinity-purification of PECAM-1 according to the presentinvention. These Mabs include anti-hec7, identified byr Pluller, et al.,J. Exp. Med. 170: 399-414 (1989); SG134, disclosed by Goyert, et al., J.Immunol. 137: 3909 (1986); CLB-HEC 75, disclosed by van Mourik, et al.,J. Biol. Chem. 260: 11300 (1985); and TM2 and TM3, identified by Ohto,et al., Blood 66: 873 (1985).

As an alternative to immunoaffinity chromatography, conventionalglycoprotein purification employing column chromatography can also beemployed in isolating PECAM-1 from cellular sources. See Ausubel supra,at Sections 10.12-10.15. A PECAM-1 isolation by this route would involvethe use of one or more of ion-exchange high pressure liquidchromatography (HPLC), size-exclusion (SE)-HPLC, high performancechromatofocusing and hydrophobic-interaction chromatography. Highperformance chromatofocusing and hydrophobic-interaction chromatographywould be preferred isolation means since both methods provide rapidpurification of biologically active, detergent-soluble proteins likePECAM-1, with minimal denaturation and high recovery.

Based on the nucleotide sequence of FIG. 1 and knowledge of theextracellular, transmembrane and intracellular domains of the maturePECAM-1 molecule (see FIG. 2), polypeptide molecules can also beproduced which represent variations of the naturally occurring molecule.These polypeptide molecules are referred to here generically as "PECAMvariants" and include, for example, PECAM muteins and molecules thatcorrespond tco portions of PECAM-1.

In this regard, a "PECAM mutein" is a polypeptide that retains the basicstructural attribute--namely, the immunoglobulin homology unit--andadhesion-promoting activity of a CAM, and that is homologous to PECAM-1.For purposes of this description, "homology" between two sequencesconnotes a likeness short of identity indicative of a derivation of thefirst sequence from the second. in particular, a polypeptide is"homologous" to PECAM-1 if a comparison of amino-acid sequences betweenthe polypeptide and PECAM-1, reveals an identity of greater than about70%. Such a sequence comparison can be performed via known algorithms,such as the one described by Lipman and Pearson, Science 227: 1435(1985), which are readily implemented by computer.

PECAM muteins can be produced, in accordance with the present invention,by conventional site-directed mutagenesis, which is one avenue forroutinely identifying residues of the PECAM-1 molecule that can bemodified without rendering the resulting polypeptide biologicallyinactive. See, Ausubel supra, at Section 8. Oligonucleotide-directedmutagenesis, comprising [i] synthesis of an oligonucleotide with asequence that contains the desired nucleotide substitution (mutation),[ii] hybridizing the oligonucleotide to a template comprising astructural sequence coding for PECAM-1 and [iii] using T4 DNApolynterase to extend the oligonucleotide as a primer, is preferredbecause of its ready utility in determining the effects of particularchanges to the PECAM-1 structural sequence. Its relative expense maymilitate in favor of an alternative, known direct-mutagenesis method.

Also exemplary of PECAM variants within the present invention aremolecules that correspond to a portion of PECAM-1, or that comprise aportion of PECAM-1 without being coincident with the natural molecule,and that display the adhesion-promoting activity of a CAM, as describedabove. Among these variants would be a polypeptide containing anamino-acid sequence that corresponds to the extracellular domain ofPECAM-1, absent the transmembrane or intracellular portions; that is, toa "soluble receptor" form of PECAM-1.

Other PECAM variants of the present invention are fragments of PECAM-1that retain CAM-like adhesion-promoting activity. Likewise within thepresent invention would be synthetic polypeptides that (i) correspond toa portion of the PECAM-1 amino-acid sequence and (ii) retain an activitycharacterisitic of PECAM-1. Such synthetic polypeptides would preferablybe between 4 and 100 amino residues in length.

Whether a synthetic polypeptide meeting criterion (i) also satisfiescriterion (ii) can be routinely determined by means of an assay for aPECAM-1 activity. Two such activities are the mediation, respectively,of adhesion-dependent movement of cells in response to a chemicalattractant (chemotaxis) and of endothelial cell--cell adhesion. Theformer activity can be assayed, following Ohto, et al., Blood 66: 873-81(1985), by determining whether an antibody that binds the syntheticpolypeptide in question also inhibits chemotaxis of neutrophils ormonocytes responding to a suitable stimulant, such as E. coli endotoxin.The ability to mediate adhesion between endothelial cells can besimilarly assayed in routine fashion, i.e., by testing whether apolypeptide-recognizing antibody inhibits the anchoring of dispersedendothelial cells to an suitable substrate and/or the formationthereafter of intercellular junctions.

PECAM variants could be produced by known de novo-synthesis techniquesand by fragmentation of the PECAM-1 molecule itself, as well as byproducing a genetically-engineered host cell that expresses a PECAMfragment encoded by a heterologous polynucleotide used to transform thehost. To be used in recombinant expression of a PECAM-1 or a PECAMvariant, a polynucleotide molecule encoding PECAM-1 or a PECAM variantwould preferably comprise a nucleotide sequence, corresponding to thedesired amino-acid sequence, that is optimized for the host of choice(see below) in terms of codon usage, initiation of translation, mostsuitable glycosylation, and expression of commercially useful amounts ofPECAM-1 or a desired PECAM variant. Also, the vector selected fortransforming the chosen host organism with such a polynucleotidemolecule should allow for efficient maintenance and transcription of thesequence encoding the polypeptide.

Preferred among the hosts generally available for transformation,pursuant to the present invention, are eukaryotes capable of effectingglycosylation at sites identified in FIG. 1. These eukaryotes areexemplified by yeast expression systems, including those employingspecies of Saccharomyces, Pichia and Kluyveromyces, respectively. See,for example, U.S. Pat. No. 4,456,082 and No. 4,837,147 (Saccharomyces);U.S. Pat. No. 4,855,231, No. 4,808,537 and No. 4,857,467 (Pichia); andU.S. Pat. No. 4,806,472 and No. 4,859,596 (Kluyveromyces).

The choice of an appropriate yeast strain for transformation will bedetermined in large part by the selectable marker(s) and other featuresof the vector employed. In addition, since a recombinant PECAM moleculeslated for therapeutic application may elicit an allergic reaction tohost-related carbohydrate moieties with which the molecule isglycosylated, it may prove advantageous to select a host for the abilityto produce a heterologous PECAM protein having reduced glycosylation.Once such a PECAM-producing host cell has been selected, then specificclones can be screened to select for variations in glycolsylationpattern that are most suitable for a therapeutic application. Yeaststrains which are exemplary of those suitable for use according to thepresent invention are strain X2181-1B, with genotype α trp1 gal1 ade1his2 (available from the Yeast Genetic Stock Center, Berkeley,California); strain ATCC 52683, with genotype α his2 ade1 trp1 met14 ura3 (aka strain "J17," available from the American Type CultureCollection, Rockville, Md.); and strain ATCC 46183, with genotype α his1trp1 (aka "strain IL166-5B," also available for the American TypeCulture Collection).

Another type of preferred expression system entails the use of amammalian host cells transformed with a polynucleotide within thepresent invention. Illustrative of suitable mammalian-cellular hostswhich can be used for this purpose are Chinese ovary (CHO) cells asdescribed by Urlaub & Chasin, Proc. Nat'l Acad. Sci. USA 77: 4216(1980); baby hamster kidney (BHK) cells, exemplified by a cell linedeposited under accession number ATCC CCL 10 and another line ATCC CCL70; monkey kidney CV1 cells transformed with SV40, exemplified by a linedeposited under accession number ATCC CRL 1651 (aka "COS-7"); humanembryonic kidney cells of the sort represented in a cell line describedby Graham, et al., J. Gen. Virol. 36: 59 (1977); mouse sertoli cells asdescribed by Mather, Biol. Reprod. 23: 243-51 (1980); african greenmonkey kidney cells as exemplified by line ATCC CRL 1587 (aka"VERO-76"); human cervical carcinoma (HELA) cells (see, e.g., line ATCCCCL 2); canine kidney cells of the sort deposited under ATCC 34 (aka"MDCK"); buffalo rat liver cells as exemplified by line ATCC CRL 1442(aka "BRL 3A"); human lung cells such as are represented by line ATCCCCL 75 (aka "W138"); human liver cells (see, e.g., Hep G2, HB 8065);mouse mammary tumor cells of the sort deposited under ATCC CCL 51 (aka"MMT 060562"); and TRI cells as described by Mather, et al., Ann. N.Y.Acad. Sci. 383: 44 (1982).

For both yeast and mammalian expression systems, there are conventionaltransformation and screening protocols which can be employed pursuant tothe present invention. Standard methodology in this regard is detailedin CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Chapter 9 (mammalian cells)and Chapter 13 (yeast cells), loc. cit. For example, transformants canbe selected for expression of functional PECAM-1 or a PECAM variant byuse of MAbs which display an appropriate PECAM specificity, as describedabove.

For introducing a polynucleotide of the present invention into a yeastcell, the most commonly used protocol, the lithium acetate method,exploits the fact that alkali cations make yeast cell membrane permeableto DNA; in addition, uptake of foreign DNA is promoted by the presencein the medium of a high-molecular-weight molecule, polyethylene glycol.An alternative method, spheroplast transformation, can be used but ismore time-consuming than the lithium acetate procedure but results in ahigher efficiency of transformation per input DNA.

Introduction of a polynucleotide of the present invention into mammaliancells to produce a recombinant cell which expresses PECAM-1 or a PECAMvariant can be accomplished according to conventional procedures, suchas by calcium phosphate or DEAE-dextrane transfection. See, e.g.,Ausubel supra, at Chapter 9. Expression of such recombinant cells of thepresent invention provides recoverable amounts of a PECAM-1 or a PECAMvariant.

As discussed below, PECAM-1 is at least immunologically related to anantigen, designated "CD31," that has been characterized serologically byLee, et al., J. Exp. Med. 168: 1193-98 (1988), but never provided inrecoverable form. CD31 displays differential expression in various typesof leukemia and, hence, can be used as a indicator of leukemia type.PECAM-1 should likewise be useful as a leukemia indicator, and labeledoligonucleotide probes ("PECAM probes") can be designed, based on apolynucleotide encoding PECAM-1 or a PECAM variant, that should find usein the classification of various leukemic states.

A PECAM probe of this sort can be a fragment of an oligonucleotide thatis complementary to a polynucleotide encoding PECAM-1 or a PECAMvariant. Alternatively, a synthetic oligonucleotide can be used as aPECAM probe which is preferably at least about ten nucleotides inlength, in order to be specific for a PECAM-1- or PECAM variant-encodingpolynucleotide. Nucleic acids can be isolated from the blood of apatient or from a tissue biopsy and analyzed by hybridization tolabelled PECAM probes. Such probes preferably hybridize toPECAM-1-encoding nucleotides under high stringency conditions, i.e.,under hybridization and washing conditions (buffer ionic strength,temperature, duration, etc.) such that nonspecific binding of a probe isminimized.

For probe design, hybridization technique and stringency conditions, seeAusubel supra, sections 6.3 and 6.4. Additional approaches to probedesign and detection can also be used, e.g., ligase-mediated genedetection (LMGD), as disclosed by Landegren, et al., Science 24: 1077-80(1988) and fluorescence resonance energy transfer (FRET), as disclosedby Wolf, et al, Proc. Nat'l Acad. Sci. USA 85: 8790-94 (1988).

PECAM-1 is also believed to play a role in chemotaxis by neutrophilcells and in the formation of endothelial-cell intercellular junctions.PECAM-1 or a PECAM variant, especially the soluble-receptor formdescribed above, should therefore prove useful in modulating angiogenicprocesses which depend on neutrophil chemotaxis and/or formation ofjunctions between enidothelial cells, for example, in tumor development.

In a related vein, so-called "antisense" oligonucleotides can beprepared as polynucleotides complementary to (a) nucleotide sequencescomprising a DNA which codes for PECAM-1 or a PECAM variant or (b)nucleotide sequences comprising PECAM-1 or PECAM variant messenger RNA(mRNA). For both types, the length of an antisense oligonacleotide ofthe present invention is not critical so long as there is no promotersequence (for DNA) or Shine-Delgarno site (for RNA) present. Type (a)antisense oligonucleotides would be synthesized de novo, for example,based on knowledge concerning the nucleotide sequence of the cDNA insertshown in FIG. 1. Type (b) antisense oligonucleotides could also beproduced de novo (DNA or RNA) or by transforming an appropriate hostorganism with DNA that is transcribed constitutively into RNA whichbinds a PECAM-1 or PECAM variant mRNA. Both type (a) and type (b)oligonucleotidee within the present invention are expected to be usefulas agents for "down-regulating" (turning off) the expression of PECAM-1on the cell surface, or inhibiting either transcription [type (a)] ortranslation [type (b)].

PECAM-1 and PECAM variants, or antibodies raised against one or more oftheise polypeptides, are expected to have a utility in the prevention ofmetastatic disease. In this context, the term "antibody" encompassesmonoclonal and polyclonal antibodies. Such an antibody can belong to anyantibody class (IgG, IgM, IgA, etc.). For monoclonal antibody (Mab)production, one generally proceeds by isolating lymphocytes and fusingthem with myeloma cells, producing hybridomas. The cloned hybridomas arethen screened for production of "anti-PECAM" antibodies, i.e.,antibodies that bind preferentially to either PECAM-1 (full-length ormature form) or a PECAM variant. "Antibody" also encompasses fragments,like Fab and F(ab')₂, of anti-PECAM antibodies, and conjugates of suchfragments, and so-called "antigen binding proteins" (single-chainantibodies) which are based on anti-PECAM antibodies, in accordance, forexample, with U.S. Pat. No. 4,704,692.

Alternatively, Mabs or a fragment thereof within the present inventioncan be produced using conventional procedures via the expression ofisolated DNA which codes for variable regions of such an Mab in hostcells like E. coli, see Ward, et al., Nature 341: 544-46 (1989), ortransfected murine myeloma cells, see Gillies, et al., Biotechnol. 7:799-804 (1989), and Nakatani, et al., loc. cit., 805-10. In addition,Fab molecules can be expressed and assembled in a geneticallytransformed host like E. coli. A lambda vector system is available thusto express a population of Fab's with a potential diversity equal to orexceeding that of subject generating the predecessor antibody. See Huse,et al., Science 246: 1275-81 (1989).

Antibodies against PECAM-1 or a PECAM variant can also be employed inthe generation, via conventional methodology, of anti-idiotypicantibodies (antibodies that bind an anti-PECAM antibody), e.g., by theuse of hybridomas as described above. See, for example, U.S. Pat. No.4,699,880. Such anti-idiotypic antibodies could be used to sequesteranti-PECAM antibodies in an individual, thereby to treat or preventpathological conditions which may be associated with an immune responsewhereby PECAM-1 is recognized as "foreign" by the immune system of theindividual.

The present invention is further described by reference to thefollowing, illustrative examples.

EXAMPLE 1

Preparation of Enriched Lysate Containing PECAM-1, and Generation ofMultispecific Polyclonal Antibodies to Platelet Membrane Proteins.

A lysate enriched for human platelet integral membrane proteins wasprepared in accordance with Newman, et al., Thrombosis Res. 27: 221-24(1982), and multispecific polyclonal antibody directed against thelysate was prepared, following Newman, et al., J. Cell Biol. 103: 81-36(1986). Thus, platelet concentrates were obtained by differentialcentrifugation of units of whole human blood, and platelet membraneswere prepared from the concentrates by sonication and differentialcentrifugation. See Newman, et al., J. Cell Biol. 90: 249-53 (1981). Oneunit of platelet concentrate yielding a membrane pellet which contained5-8 mg of protein. The pellet was dissolved in 2 ml of 1% Triton X-114(Sigma Chemical Company, St. Louis, Mo.) in 10 mM Tris, 150 mM NaCl (pH7.4), which contained both 0.4 mM phenylmethylsulfonyl fluoride and 5 mMEDTA as protease inhibitors. After brief sonication to disperse membraneaggregates, solubilization was carried out on ice for 60 minutes.

The Triton-solubilized membranes were then centrifuged at 100,000×g for60 minutes, and 200 μl of the supernatant carefully applied to the topof a 300 μl of 6% sucrose cushion in a 1-ml polystyrene Fishercentrifuge tubes (Fisher Model 59 microcentrifuge). The tube was warmedabove the cloud point of the detergent by incubation for five minutes at37° C., and then centrifuged for five minutes at 1500×g to pellet thelarge protein-detergent mixed micelle aggregates which formed. Theaqueous phase, which remained on top of the sucrose cushion, wasremoved, cooled to 0° C. and re-extracted by the addition of ice-coldTriton X-114 to a final concentration of 1%. This solution was againwarmed, loaded back on top of the 6% sucrose cushion, and centrifuged asabove. The detergent phase was recovered as a yellowish oily dropletbeneath the sucrose cushion.

Both the detergent and aqueous phases were first assayed for proteinconcentration (3) and then diluted 1:1 with SDS solubilization solution(4% SDS, 100 mM Tris-HCl, pH 6.8, 10% glycerol, 0.001% bromphenol blue)prior to analysis by SDS-polyacrylamide gel electrophoresis. For reducedsamples, 2-mercaptoethanol was added to a final concentration of 5%.After boiling the samples for five minutes, 5-50 ug/well of protein wasapplied to a 7% SDS polyacrylamide slab gel and electrophoresed forapproximately 3 hours at 30 mA. After electrophoresis, gels were fixedand silver stained according to the method of Merril, et al., Science211:1437-38 (1981).

The polyclonal antiserum was prepared in rabbits by twice-monthlyintradermal injection of the enriched lysate described above. PurifiedIgG was obtained using a conventional combination of ammonium sulfatefractionation and DEAE cellulose chromatography. The specificity of theresulting preparation was confirmed by immiunoprecipitation ofradioiodinated, solubilized platelets, by crossed immunoelectrophoresisvs. solubilized whole platelets, and by western blot analysis, accordingto known methods. See, e.g., Ausubel supra, at Section 10.

EXAMPLE 2

Antibody Screening of cDNA Library for PECAM-1-Encoding Clones, andEpitope Selection of PECAM-1-Specific Antibody.

The polyclonal antiserum thus prepared was then used to screen an cDNAλgt11 expression library made from the permanent endlothelial cell/lungcarcinoma hybrid cell line, EA.hy 296, described by Edgel, et al., Proc.Nat'l Acad. Sci. USA 80: 3734 (1983). In particular, the identities ofantibody-positive clones were determined using the "epitope selection"method disclosed, e.g., by Hall, et al., Nature (London) 311: 379(1984), in which immobilized β-galactosidase fusion proteins fromplaque-purified, antibody-positive clones were used to select from thepolyspecific antiserium those antibodies capable of binding specificepitopes encoded by each fusion protein.

By this approach two clones, 8B and 8C, were identified that selectedantibodies reacting on immunoblots with a single, 130-kd plateletprotein. To isolate the 130-kd protein from the known platelet membraneglycoproteins in the same molecular-weight range, two-dimensionalnonreduced/reduced gel electrophoresis was employed to permit accuratecharacterization of the protein encoded by clones 8B and 8C.

More specifically, the λgt11 bacteriophage clone 8B, containing a 1.6 kbinsert, was grown on a lawn of E. coli strain 1090 to a density ofapproximately 50,000 pfu/plate and induced for expression of itsβ-galactosidase fusion protein with isopropylthiogalactoside. The platewas overlayed with a 150 mm nitrocellulose circle for 3 hours to adsorbthe induced phage proteins. After blocking unbound sites with gelatin,the nitrocellulose membrane was incubated with a polyclonal rabbitantiserum raised against human platelet integral membrane proteins,thereby allowing the antibody population specific for the expressedepitopes to specifically bind to the immobilized fusion protein. Afterextensive washing, the epitope-selected antibody, ES8B, was eluted fromthe membrane with pH 2.3 glycine-HCl, neutralized with Tris base, andreacted with a western blot of whole platelet lysates to demonstrate aspecificity by the antibody for the unique protein spot representing the130-kd platelet protein. Binding was visualized by means of an alkalinephosphatase-conjugated second antibody followed by a5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium saltsubstrate pair.

EXAMPLE 3

PECAM-1 Characterization and Binding by Epitope-Selected Antibody.

The epitope-selected antibody from clone 8B thus reacted on western blotwith PECAM-1 as a single protein spot that was distinct from otherplatelet membrane proteins, including GPIa, GPIIa, GPIIb and GPIIIa. Theprotein distinguished in this manner, PECAM-1, migrated slightly above adiagonal line formed by proteins for which electrophoretic migration isunaffected by reducing agents, indicating that the isolated protein is asingle-chain protein containing intrachain disulfide bonds.

Various biochemical criteria found applicable to PECAM-1 are consistentwith a protein that is a highly glycosylated, major surface membraneglycoprotein. Thus, treatment of intact platelets with trypsin,neuraminidase and N-glycanase, respectively, caused a marked reductionin the molecular weight of isolated PECAM-1 polypeptide, as assessed bywestern blot analysis of subsequently washed and detergent-lysedplatelets. This reduction in molecular weight indicated thatglycosylation of PECAM-1 was significant and that the protein portioncontained exposed amino-acid sequences that were susceptible to trypsinhydrolysis. PECAM-1 was labeled effectively by treatment of intactplatelets with periodate/sodium boro[³ H] hydride, acarbohydrate-specific label, and less so by lactoperoxidase-catalyzedsurface radioiodination. Semiquantitative, comparative western blotanalysis, effected by means of antibody probes to a number of different,well-characterized platelet membrane glycoproteins indicated thatPECAM-1 is a major membrane component. That PECAM-1 stains only weaklywith Coomassie blue accounts for its not having been isolated andcharacterized previously.

A series of monoclonal antibodies previously raised against platelet andendothelial cell surface glycoproteins similar in size and pI to PECAM-1were tested for their ability to react with the latter molecule. Two ofthese monoclonal antibodies, anti-CD31 and anti-hec7, reacted with aplatelet protein the co-migrated on SDS-PAGE gels with PECAM-1, Aspreviously mentioned, CD31 is a leukocyte differentiation antigen ofuncharacterized structure and function which has been reported to bepresent on (but has never been obtained in recoverable amounts from)human platelets, enidothelial cells, granulocytes and monocytes. seeKnapp, et al., Immunology Today 10: 253 (1989). The hec7 antigen, whichhas also not been isolated in recoverable amounts, is reported to belocalized to the intercellular junctions of human endothelium. Muller,et al., J. Exp. Med. 170: 399 (1989).

Preparations containing the hec7 and CD31 antigens were prepared, viaimmunoprecipitation with their respective monoclonal antibodies, fromhuman endothelial cell lysates. (MAbs provided by Dr. William A. Muller(Rockefeller University) and Dr. Sanna M. Goyert (Cornell University).)Analysis of the preparations by immunoblotting with an anti-PECAMantiserum indicated that PE:CAM-1, the CD31 antigen and the hec7 antigenare at least immunologically related.

EXAMPLE 4

Determination of a Nucleotide Sequence Coding for PECAM-1.

The 1.6 kb cDNA insert from clone 8B was subcloned into a pUC18-derivedplasmid vector, pTZ18r. The 8B insert was then used as a probe accordingto known procedures, as disclosed, for example, by Ausubel supra, atSection 6.3, to identify twenty-three additional clones which weresubsequently characterized by restriction mapping and hybridizationanalysis according to known methods. Two of these clones, 6-4 (spanningnucleotide 1 to about nucleotidte 1170 in FIG. 1) and 6-2 (fromnucleotide 715 to nucleotide 2557), were found to overlap and containthe entire PECAM-1 encoding sequence, depicted in FIG. 1.

As mentioned above, the 2557-bp PECAM-1 sequence includes a 141-bp 5'untranslated (UT) region and a 3' UT region of 202 bp, in addition tothe 2214-bp coding sequence for the mature protein plus signal peptide.The 5' UT. region contains two eukaryotic initiation (AUG) codons,beginning at base 95 and nucleotide 142, respectively. Both of thesecodons are flanked by nucleotides, constituting a "Kozak sequence" thatshould accommodate initiation of protein synthesis by virtue of itsbeing largely devoid of T residues and having a purine at the -3position. See Kozak, et al., Nucleic Acids Res. 15: 8125 (1987).

As is true for many upstream initiation codons lying within a Kozaksequence, the ATG at base 95 of the PECAM-1-encoding nucleotide sequenceis followed by a TAA stop codon only 15 bases downstream. Thisarrangement indicates that translation likely is effected for the 15-bp"minicistron" and then is terminated, after which ribosomes re-initiatetranslation at base 142 and continue until the stop codon at base 2356.

EXAMPLE 5

Purification of PECAM-1 from a Natural Source.

Recoverable amounts of substantially pure PECAM-1 were isolated fromblood platelets as follows. Platelets were prepared from whole blood bydifferential centrifugation according to conventional procedures.Platelets were washed several times in TBS (20 mM TRIS, 150 mM NaCl, 1mM EDTA, pH 7.4) and then solubilized in TBS containing 1% Triton X-100and lmM CaCl₂ in the presence of protease inhibitors (0.4 mMphenylmethylsulfonyl chloride, 10 μM leupeptin). The mixture was nextclarified by centrifugation at 15,000 g for 30 minutes, and thesupernatant WCLS then added to a column of Conconavilin A sepitarose toremove any other glycoproteins. The flow-through of this column wasapplied to an immunoaffinity column specific for PECAM-1, usinganti-hec7 antibody attached to a sepharose column. PECAM-1 was theneluted by a brief pulse of a solution containing 100 mM glycine/HCL and0.1% Triton X-100 at pH 2.4.

The eluate was neutralized with TRIS base and analyzed by SDS-PAGE toestablish purity. If further purification was necessary, then the eluatewas adsorbed to a wheat germ sepharose column and eluted off with 0.3MN-acetylglucosamine. Thus, PECAM-1 was obtained, in recoverable amounts,in a preparation that ran as a single band on both a reducing and anon-reducing SDS-PAGE gel.

What is claimed is:
 1. An isolated polynucleotide encoding the mature form of platelet-endothelial cell adhesion molecule-1 (PECAM-1) having the amino acid sequence depicted in FIG. 1, amino acids 1 to
 711. 2. The isolated polynucleotide of claim 1, wherein said polynucleotide consists of the nucleotide sequence depicted in FIG. 1, nucleotides 222 to
 2355. 3. An isolated polynucleotide encoding a polypeptide consisting of the signal sequence of platelet-endothelial cell adhesion molecule-1 (PECAM-1) and PECAM-1, wherein said polypeptide has the amino acid sequence depicted in FIG. 1, amino acids -27 to
 711. 4. The isolated polynucleotide of claim 3, wherein said polynucleotide comprises the nucleotide sequence depicted in FIG. 1, nucleotides 141 to
 2355. 5. The isolated polynucleotide of claim 4, wherein said polynucleotide has the nucleotide sequence depicted in FIG. 1, nucleotides 1 to
 2557. 6. An isolated polynucleotide encoding the extracellular domain of platelet-endothelial cell adhesion molecule-1 (PECAM-1) having the amino acid sequence depicted in FIG. 1, amino acids 1 to
 574. 7. The isolated polynucleotide of claim 6, wherein said polynucleotide consists of the nucleotide sequence depicted in FIG. 1, nucleotides 222 to
 1945. 8. An isolated polynucleotide encoding a polypeptide consisting of the signal sequence of platelet-endothelial cell adhesion molecule-1 (PECAM-1) and the extracellular domain of PECAM-1, wherein said polypeptide has the amino acid sequence depicted in FIG. 1, amino acids -27 to
 574. 9. The isolated polynucleotide of claim 8, wherein said polynucleotide consists of the nucleotide sequence depicted in FIG. 1, nucleotides 141 to
 1945. 10. A plasmid comprising the polynucleotide of claim
 7. 